Synthesis and In Vitro Characterization of L-ornithine Conjugated Poloxamer P407

Abstract

L-ornithine (ORN) is a naturally occurring amino acid involved in the urea cycle. It is the substrate of the ornithine decarboxylase (ODC) enzyme. ODC converts ORN to putrescene and C02. Studies have shown that ODC is overexpressed in several types of cancer. So, ORN can be used as a ligand for ODC-targeted delivery to cancer tissues. Poloxamer P407 (PLX) has been proved as a safe polymer with dual properties of micellization and thermos-gelling, which can effectively be used for cancer-targeted therapies. This study aims to conjugate PLX with ORN and check the impact of conjugation on the intrinsic properties of PLX. For this purpose, the amide bond is formed between PLX and ORN using 1,1 - carbonyl diimidazole (CD I) as a carboxylic group activator under the nitrogen environment at room temperature. The synthesis of L-ornithine conjugated poloxamer P407 (PLX-ORN) was confirmed using FTIR analysis. Critical micelle concentration (CMC) ofPLX and PLX-ORN at 25°C was 3.8 gIL and 3.4 gIL respectively and at 37°C it was 0.07 gIL and 0.04 gIL. The critical micelle temperature (CMT) was determined at three different concentrations i.e., 189 gIL, 2.82 giL, and 0.064 gIL. For PLX the CMT was 7.5°C, 23.3°C, and 31°C respectively and for PLX-ORN it was 5°C, 21.8°C, and 27.8°C respectively. CMC was slightly affected by the addition of normal saline. Gelation properties were analyzed which indicated a decrease in gelation temperature and time. Micelles from both PLX and PLX-ORN were prepared. PLX-ORN micelles had a size of217.7 nm in contrast with PLX micelles having a size of 206.0 nm. The charge ofPLX-ORN and PLX was -26.2 mV and -18.0 mY, respectively. The results indicate a decrease in the CMC and CMT in PLX-ORN as compared to PLX while there is no significant change in the size and zeta potential of the micelles of PLX-ORN and PLX. The studies concluded that ORN was successfully conjugated to PLX. The in vitro characterization indicated a decrease in CMC, CMT, CGT, and gelation time. Normal saline has a slightly decreasing effect on CMC due to the conjugation ofORN to PTX while hemolytic assay indicated the safe nature ofPLX-ORN. There was no significant effect of conjugation observed on micellization.